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Education: Electrical Stimulation

A novel tool in regenerative medicine?

Construction and Use of an Electrical Stimulation Chamber

for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

Bioelectricity: Electrical phenomena in living organisms

One might think of electricity and life as two separate phenomena that do not coexist in harmony. When one imagines being hit by lightning or getting electrocuted – injury or discomfort come to mind, not normal everyday life-as-usual! In fact, bioelectricity, like water or sunlight is an essential part of all living things. Cells use bioelectricity as signals to regulate and maintain normal functions essential to their survival like division, migration, differentiation, growth, repair and death.

Examples of normal bioelectrical function in nature include some types of fish like the Electric eel (Electrophorus electricus) and the Elephantnose fish (Gnathonemus petersii) that emit electric shocks, generated by specialized electric organs, to scan their environment to navigate and hunt for pray.

Examples in humans are less dramatic but just as essential to our survival. In fact every aspect of our daily lives requires bioelectricity. As you read this text, cells in your nerves are constantly creating and sending bioelectrical impulses – so-called action potentials – along complex networks of axons, transforming written text on your computer screen into meaningful concepts. These bioelectric impulses emitted from the brain or along peripheral nerves/muscles can be detected and measured on the surface of our skin overlaying our brain or muscles using electroencephalography (EEG) or electromyography (EMG) respectively.

What is electrical stimulation all about?

There are four basic kinds of electrical current:

Free flow of electrons through a metal conductor, like copper wire, is the most common. The body does not have this kind of electrical current.

Ionic current, as in batteries, is also very common. Ions are atoms or molecules in water, which have either a surplus or deficit of electrons. Because the ionic molecules are so big, they can't go very far. For example, an ion can move across a cell membrane (with encouragement) but it could never move the length of a neuron. In other words, although ionic currents exist in the body, they are of little impact (with one or two exceptions).

Semi-conduction was discovered in the 1930's, it was found that electrons could hop through a crystal, and give a small electric current. The body is a natural semi-conductor. The semi-conducting currents in crystals can go a very long distance, as in fiber-optic telephone wires, and can carry lots of information, as in computer chips. The first person to suggest that semi-conducting currents might be found in the body was Albert Szent-Gyorgi, who won a Nobel Prize for his work on vitamin C.

Super-conduction, in which the electric current flows without resistance, without loss of energy, was first discovered in Holland in 1911, when K. Onnes brought the temperature of mercury close to absolute zero, the lowest degree possible, and found no electrical resistance. (From: The Bio-Energy Revolution by WS Eidelman)

In a multicellular organism composed of many different organs and tissues such as muscle, cartilage, bone, vessels, epithelium, blood – cells are the individual building blocks. The individual cells that make up tissues and organs must communicate with the cells in their immediate proximity and with distant cells in a coordinated fashion to maintain an organism’s homeostasis.

For a long time it was believed that cell-to-cell communication only occured through biochemical reactions. More recently, however we have learned that in conjunction with these biochemical signals electric signals play a key role in this cell-to-cell communication (see Levin et al. 2012 Molecular bioelectricity). Electrical signals are generated within cells, across cells and across whole tissues. These steady, trans-cellular and trans-tissue potentials play an important role in every aspect of an organism’s day-to-day function and in the activities that are essential for its existence and survival - development, growth, repair, etc...

We can study bioelectricity in individual cells and cell colonies in in-vitro cell cultures by applying voltages, in special chambers, and measuring how they respond.

"Application of direct current electricfields to cells and tissues grown inculture."

Using this setup we can learn how electrical fields effect cell migration, proliferation, changes in morphology and gene expression. By varying the electrical and chemical conditions in the culture medium in these in-vitro systems we can learn and estimate how bioelectricity behaves in living organisms.

The History of our Knowledge of Bioelectricity

The study of Electrophysiology and Bioelectricity has a very long history. The work of Luigi Galvani (1737–1798) and Alessandro Volta (1745–1827) paved the way for many different experiments on electricity in medical science. However, in its early days bioelectricity was discredited as charlatanry due to many bogus therapies that were developed. For example, in the 19th century, the development of an electric air bath (negative breeze) falsely claimed that it healed several diseases.

The electric air bath with a metal cathode above the patient’s headand a static electricity generator(metal plate) under the patient’sfeet promised the healing of manydiseases.

It was not until recently that bioelectricity was re-discovered and gradually re-embraced by the scientific community, based on rigorous research. This research has begun to revile the role bioelectricity plays in normal tissue development, growth, repair and disease. Recent studies have demonstrated that in early stages of embryogenesis such as the neurula stage electrical fields exist throughout the tissue of the embryo and play a key role in normal development.

By short-circuiting normally occurring electrical fields in the developing chick embryo important developmental deformities resulted, demonstrating the critical importance of bioelectricity at this critical early stage of development. These experiments demonstrate the role electrical currents play in guiding cell movement and establishing basic developmental patterns in the developing chick.

Bioelectricity in Wound Healing

Several lines of research have demonstrated the role of bioelectricity in wound healing.In 1984 the German (electro-) physiologist Emil Du-Bois Reymond was able to measure what he called an “injury potential” of about 1µA from a cut he made on his own finger.

A cut in the skin disrupts the natural flow of electrical currents resulting in it leaking from the dermal wound.

This leakage of electrical current sends signals to the surrounding tissues and plays a key, and still poorly understood, role in wound healing. The molecular basis for this injury potential results from different concentrations of ions (especially sodium and chloride) on both sides of a tight web of tight junctions (McCaig, Rajnicek, et al. 2005), which prevent the short circuit.

Bioelectricity and Limb Regeneration

Electrical currents across cells play a key role in vertebrate regeneration. It is known that there are surface potential differences between various parts of the adult salamander. In 1941 Monroy observed that the distal end of a salamander’s limb is positive compared to its proximal end. He went on to show that amputating the limb significantly increased this difference. Further experiments demonstrated that current leaving the cut surface of the regenerating limb stump reentered proximal to this point. Before amputation, current enters the skin from all parts of the limb’s skin surface, after amputation, there is an increase in the density and intensity of the current emitting from the surface of the amputation stump. (From: Bioelectricity and Regeneration, By RB Borgens, et al. BioScience, 29(8):468-74 (1979)

Pattern of current around (A) intact newt forelimb and (B) forelimb stump after amputation.Arrows indicate direction of current and magnitudeof current density, as measured 0.3 mm from thesurface. Note the difference between the scales inA and B to allow visualization of the relatively smallcurrent densities of intact skin.

These large currents emitting from the amputated stump persist for about 10-14 days until early blastema formation and then as the limb grows decrease to pre-amputation levels. These findings clearly demonstrate that bioelectricity is involved in salamander limb regeneration. (From: Bioelectricity and Regeneration, By RB Borgens, et al. BioScience, 29(8):468-74 (1979)

Bioelectricity and Limb Regeneration in Mammals

The above mentioned experiments, measuring electrical currents in regenerating limbs, were performed in animals (salamanders) that naturally regenerate their limbs in nature. Based on these observations scientists began to test the effect electrical currents from an external source had on limb regeneration. They performed these experiments in salamanders and frogs that regenerated their limbs naturally, and in rats, that do not. What they found was an important discovery at the time and surprised the scientific community. They demonstrated that externally delivered electrical current stimulated the initiation of limb regeneration (blastema formation) in rats, that in normal conditions do not regenerate their limbs! These experiments were performed in several different laboratories (Becker – 1972, Smith – 1981, Libbin – 1979, Siskin -1979) confirming these findings.